A proper balance between the activities of coagulation and fibrinolytic cascades is needed both to protect an organism from excessive blood loss upon injury and to maintain blood flow within the vascular system. The two opposing coagulation and fibrinolytic cascades are recognized to comprise a series of zymogen to enzyme conversions which termninate in the two respective proteolytic enzymes, thrombin and plasmin. These enzymes catalyze the formation and removal of fibrin within the circulatory system. Imbalances are characterized by either bleeding or thrombotic tendencies which may result in heart attacks or strokes in the organism.
Thrombin activatable fibrinolytic inhibitor (TAFI) is a 60 kD glycoprotein present in human plasma that modulates fibrinolyisis in vivo. TAFI present in plasma is a proenzyme form which is most efficiently activated by proteolytic cleavage at Arg-92 with a thrombin-thrombomodulin complex. The proenzyme form of TAFI may also be activated by proteolytic cleavage by other proteolytic enzymes including, but not limited to, thrombin or plasmin. Upon activation of the TAFI proenzyme by proteolytic cleavage with thrombin-thrombomodulin, an active enzyme of 35 kD is formed with carboxypeptidase-like activity (TAFIa). This molecule has also been referred to in the literature as plasma carboxypeptidase B (PCPB), or plasma carboxypeptidase U (PCPU). TAFIa rapidly loses enzymatic activity through a temperature-dependent (t1/2=10 min at 37° C.) inactivation process. The inactivation of TAFIa is not due to a proteolytic process, but rather to a conformational change in the protein structure which can be followed by a change in the fluorescence spectrum of the protein. The inactivated TAFIa enzyme is referred to as TAFIai (35 kD). The spontaneous temperature-dependent inactivation of TAFIa to TAFIai is believed to be the major mechanism by which TAFIa activity is modulated during fibrinolysis.
Modulation of fibrinolysis occurs when TAFIa cleaves C-terminal arginine and lysine residues of partially degraded fibrin, thereby inhibiting the stimulation of tissue plasminogen activator (t-PA) modulated plasminogen activation. The fibrinolytic system is activated primarily by t-PA which is provided by damaged cells in the blood vessel wall. t-PA converts circulating plasminogen to the active protease plasmin and can produce either slow enhancement of fibrinolysis or, when combined with fibrin, rapid enhancement of fibrinolysis. The effect of t-PA on fibrinolysis can be blocked by a class of inhibitors termed plasminogen activator inhibitors (PAIs), of which several have been identified.
Thrombomodulin is a component of the blood vessel wall which binds thrombin and changes its specificity from fibrinogen to protein C, resulting in a molecule possessing anticoagulant, rather than procoagulant, activity. The thrombin-thrombomodulin complex catalyzes cleavage of protein C to activated protein C, which results in down-regulation of the coagulation cascade by proteolytically inactivating the essential co-factors, Factor Va and VIIIa. In this manner, the body regulates coagulation cascade.
Studies such as that by Taylor et al., Thromb. Res. 37:639 (1985) have suggested that activated protein C is not only an anticoagulant, but also profibrinolytic, both in vivo and in vitro. Subsequently, it was determined that protein C only appears profibrinolytic because it prevents the thrombin-catalyzed activation of a previously unknown fibrinolysis inhibitor, whose precursor was isolated from plasma and designated as being TAFI.
TAFI was discovered independently in three different laboratories. In initially appeared as an unstable carboxypeptidase B-like molecule in human serum and was described by Hendriks et al., Biochim. Biophys. Acta 1034:86 (1990). A year later the cDNA for the molecule was cloned, its amino acid sequence was described, its activation by trypsin and its enzymatic properties toward synthetic carboxypeptidase B substrates was reported (see U.S. Pat. No. 5,206,161). In 1994, Wang et al., (J. Biol. Chem. 269:15937 (1994)) isolated the activated molecule and named it carboxypeptidase U (“U” being designated for unstable). Subsequently, Nesheim et al. (J Biol. Chem. 270:14477 (1995)) showed that the protein was both activated by thrombin and inhibits fibrinolysis, and designated the molecule TAFI. The co-identity of PCPB, PCPU, and TAFI has been established by their independent chromatographic behavior on plasminogen Sepharose® and the amino acid sequences present at the activation cleavage site.
The mechanism of TAFI inhibition of fibrinolysis can be schematically described as depicted in FIG. 1.
The conversion of TAFI to TAFIa and subsequently to TAFIai indicates an ongoing fibrinolytic process. Therefore, the presence of TAFIa and TAFIai in plasma is a marker of the fibrinolytic process. Alterations of TAFI levels have been identified in a number of pathological conditions including vascular and heart diseases, liver disease, hemophilia, cancer, leukemia and stroke. Therefore, accurate quantitation of TAFI, TAFIa and TAFIai levels in plasma provides information in the understanding and diagnosis of many diseases in which fibrinolysis plays a role.
Several ELISAs (enzyme linked immunosorbent assay) are commercially available which are designed to measure the total level of all forms of TAFI in plasma (e.g., Imuclone TAFI 873 by American Diagnostica, Inc.; TAFI ELISA by Affinity Biologicals). An ELISA has been reported which only measures the 60 kD TAFI proenzyme (Stromquist et al. Thromb. Hemost. 85:12-17 (2001)). A chromogenic assay based upon a functional assay is commercially available which only measures the 60 kD proenzyme form of TAFI (Actichrome 874 by American Diagnostica, Inc.).
The 60 kD proenzyme form of TAFI shares common antigenic epitopes with TAFIa and TAFIai. There have not been any antibodies described which have are specific for TAFIa and TAFIai but not the 60 kD proenzyme form of TAFI. Accordingly, an ELISA that specifically measures TAFIa and TAFIai in the presence of the 60 kD proenzyme form of TAFI has not been developed.
Diagnostic assays selective for TAFIa and TAFIai in the presence of the 60 kD proenzyme form of TAFI would be useful for identifying and monitoring changes in the fibrinolytic process. Such changes are indicative of diseases where the fibrinolytic process is involved, such as vascular and heart diseases, liver disease, hemophilia, cancer, leukemia and stroke. Accordingly, there is a need in the art for effective assays selective for TAFIa and TAFIai levels in the presence of the 60 kD proenzyme form of TAFI.
Citation or identification of any reference in this section is not to be construed as such reference being prior art to the present application.